Photopheresis with 5-aminolevulinic acid in patients with cutaneous T-cell lymphoma – A proof-of-concept clinical trial
The objectives of this study were to measure 5-ALA dark cytotoxicity and 5-ALA-induced PpIX production and PDT effects with 5-ALA/UVA and 8-MOP/UVA on activated T lymphocytes obtained from clinical ECP patients with CTCL or cGvHD.The extracorporeal photopheresis (ECP) technology, also known as photopheresis, is an established immune modulating treatment. It has been a subject of several studies, since its regulatory approval in the treatment of cutaneous T-cell lymphomas (CTCLs) three decades ago. ECPs remain underutilized in the clinics regardless of its potential applications. In recent times, a number of activated T lymphocytes (T cells) mediated diseases are being investigated including acute and chronic graft-versus-host disease (aGvHD and cGvHD), organ transplant rejection and certain autoimmune diseases. The ECP modality involves ex vivo treatment of isolated white blood cells (WBCs) of a patient with a photosensitizer drug [8-methoxypsoralen (8-MOP)] and its subsequent activation by ultraviolet-A (UVA) light before reinfusion back to the patient. However, 8-MOP binds to both ailing and normal cells and thus eradicates both types of cells after UVA irradiation with no selectivity. Clinically, this modality is expensive, long-lasting and produces only partial response in the majority of treated patients. The current ECP modality can be advanced by using 5-aminolevulinic acid (5-ALA) a precursor of the photosensitizer protoporphyrin IX (PpIX), that has been shown to selectively induce PpIX in activated T cells and could be an alternative for 8-MOP. ALA is a potent non-carcinogenic photosensitizer precursor and found naturally in body cells. Our new concept of ECP technology with 5-ALA and UVA irradiation has various advantages: 1) selective and effective killing of malignant/activated hyper-proliferative T-cells without affecting functions of the normal resting T-cells; 2) 5-ALA/PpIX specifically targets cell membrane structures (no DNA binding) with no risk of carcinogenesis and 3) additionally photodynamic therapy (PDT) with 5-ALA induces systemic immune response. Since, 5-ALA has officially been approved for photodiagnosis and PDT, the modification of commercially certified UVA-integrated Therakos Photopheresis system with 5-ALA instead of 8-MOP should be safe and feasible. We have employed flow cytometry with immunophenotyping technique to measure 5-ALA-induced dark cytotoxicity on human leukocytes, to determine 5-ALA-induced PpIX production in activated T cells from both diluted mononuclear cells of ECP patients and healthy donors and in undiluted buffy coat samples of ECP patients; and to compare PDT effects on CD4+ (helper T-cells), CD8+ (cytotoxic T-cells), and CD25+ (activated T-cells) with 5-ALA/UVA and 8-MOP/UVA. No dark cytotoxicity of 5-ALA on the leukocytes of ECP patients was observed at concentrations up to 10mM for an incubation of up to 20 hours. 5-ALA-induced PpIX formation was found to be significantly more in activated T cells than resting T cells in both diluted mononuclear cells and in undiluted buffy coat samples. The CD4+, CD8+, and CD25+ T cells treated with 5-ALA/UVA were eradicated more than those treated with 8-MOP/UVA. Our results suggest that 5-ALA/UVA might have the potential for improving the efficacy of ECP.
Extracorporeal photopheresis (ECP), also known as photopheresis, is an immune modulating treatment. This technology exposes isolated white blood cells to a photosensitizer drug [8-methoxypsoralen (8-MOP)] and its subsequent activation by ultraviolet-A (UVA) light.ECP is used in Norway in treating cutaneous (skin) T-cell lymphoma and chronic graft versus host (cGVHD) disease patients.The rationale behind this study is to advance the current ECP modality by using photosensitizer pro-drug known as 5-aminolevulinic acid (5-ALA) and visible blue light. ALA is a potent non-carcinogenic photosensitizer precursor found naturally in body cells that terminates the diseased white blood cells selectively. The major disadvantages of conventional ECP therapy include 1) imminent mutations of normal cells by both DNA-binding 8-MOP and UVA; 2) no selectivity in killing both ailing and normal cells and 3) expensive, enduring and only partial response in the majority of treated patients. Thus, there is an urgent requirement for non-toxic, selective, inexpensive, short duration and more effective alternative. Our new concept of ECP technology with protoporphyrin IX (PpIX) precursor such as 5-ALA and non-carcinogenic visible blue light irradiation have various advantages: 1) precise and effective photodepletion of malignant/activated hyper-proliferative T-cells without affecting functions of the normal resting T-cells; 2) ALA/PpIX specifically targets cell membrane structures (no DNA binding) with no risk of carcinogenesis and 3) induces systemic anti-tumor immunity. Since, ALA has officially been approved for photodiagnosis and photodynamic therapy, the modification of commercially certified UVA-integrated Therakos Photopheresis system with ALA instead of 8-MOP should be safe and practical. Optimizing the parameters affecting ALA-ECP: Our group has been studying and developing the PDT technology for 3 decades. Dr. Sagar Darvekar has been appointed as a Postdoctoral fellow in June 2016 for this research project and he is involved in optimizing the parameters affecting ALA-ECP. In this study we are planning to advance the conventional ECP method (which uses 8-MOP and UVA light) by using 5-ALA and visible blue light; we had to optimize different parameters such as ALA concentration and its incubation time and also irradiation time with the visible blue light. We have employed spectrofluorometry and flow cytometry to determine the optimum PpIX production in the peripheral blood mononuclear cells (PBMC) isolated from the healthy donor after treating with various concentrations of 5-ALA for various time intervals. We have used JURKAT cells (an immortalized line of human T-lymphocyte cells) as a positive control for our experiments. We have also tried to optimize above mentioned parameters by employing Cell Titer Glow luminescent cell viability assays which is a homogenous method of determining a number of viable cells in the culture based on quantitation of the adenosine triphosphate (ATP) present, an indicator of metabolically active cells. Moreover, we also tried to establish in vitro T lymphocyte stimulation/activation methods to facilitate T-cell expansion in PBMC by using phytohemagglutinin (PHA) and mitogenic or co-mitogenic antibodies (CD3 and CD28) directed towards T cell receptor. Our results suggest that PpIX formation in PBMC and Jurkat cell lines is very much dependent on ALA concentrations and also incubation time. Moreover ALA dependent PpIX formation was found to be significantly more in CD3/CD28-activated cells as compare to resting cells population. The visible blue light can efficiently induce apoptosis and necrosis in PBMC and Jurkat cell lines after ALA incubation.